Human Mycoplasma pneumoniae antigen (MP Ag) Enzyme-linked immunosorbent assay (ELISA) kit Instructions for use This reagent is for research use only. This kit is used to determine the Mycoplasma pneumoniae antigen (MP Ag) in human serum, plasma and related fluid samples. content. Experimental principle: This kit uses the double antibody sandwich method to determine the level of human Mycoplasma pneumoniae antigen (MP Ag) in the specimen. The microplate is coated with the purified antibody to prepare a solid phase antibody, and the Mycoplasma pneumoniae antigen (MP Ag) is sequentially added to the microcapsule of the coated monoclonal antibody, and then combined with the HRP-labeled antibody to form an antibody-antigen-enzyme-labeled antibody. The complex, after thorough washing, was added to the substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with the Mycoplasma pneumoniae antigen (MP Ag) in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the content of human Mycoplasma pneumoniae antigen (MP Ag) in the sample was calculated by a standard curve. Kit composition: Kit composition 48-well configuration 96-well configuration Storage instructions 1 part 1 part sealing film 2 pieces (48) 2 pieces (96) Sealed bag 1 1 enzyme label coated plate 1 Ã— 48 1 Ã— 96 2 -8 Â° C preservation standard: 135 ng / L 0.5ml Ã— 1 bottle 0.5ml Ã— 1 bottle 2-8 Â° C preservation standard dilution 1.5ml Ã— 1 bottle 1.5ml Ã— 1 bottle 2-8 Â° C preservation enzyme standard reagent 3 ml Ã—1 bottle 6 mlÃ—1 bottle 2-8Â°C Preservation sample dilution 3 mlÃ—1 bottle 6 mlÃ—1 bottle 2-8Â°C Preservation developer A solution 3 mlÃ—1 bottle 6 mlÃ—1 bottle 2-8Â°C Preservation developer B solution 3 ml Ã— 1 bottle 6 ml Ã— 1 bottle 2-8 Â° C Preservation solution 3 ml Ã— 1 bottle 6 ml Ã— 1 bottle 2-8 Â° C Preservation concentrated washing solution (20ml Ã— 20 times) Ã— 1 bottle (20ml Ã—30 times) Ã— 1 bottle 2-8 Â° C preservation sample processing and requirements: 1. Serum: room temperature blood solidification for 10-20 minutes, centrifugation for about 20 minutes (2000-3000 rev / min). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again. 2. Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. 3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to. 4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When the components in the cells were detected, the cell suspension was diluted with PBS (pH 7.2-7.4), and the cell concentration reached about 1 million/ml. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 5. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 Â° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use. 6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 Â°C, but repeated freezing and thawing should be avoided. 7. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity. Operation steps 1. Dilution and loading of standard products: 10 wells of standard wells are placed on the enzyme labeling board, 100 Î¼l of standard is added to the first and second holes, and then added in the first and second holes. 50 Î¼l of the standard dilution, mix; then add 100 Î¼l from each of the first and second wells to the third and fourth wells, and then add 50 Î¼l of the standard dilution to the third and fourth wells. Then, 50 Î¼l of each of the third and fourth wells is discarded, and 50 Î¼l of each is added to the fifth and sixth wells, respectively, and 50 Î¼l of the standard dilution is added to the fifth and sixth wells, respectively. After mixing, 50 Î¼l from each of the fifth and sixth holes is added to the seventh and eighth holes, respectively, and then 50 Î¼l of the standard dilution is added to the seventh and eighth holes, respectively, and mixed from the seventh. 50 Î¼l of the eighth well was added to the ninth and tenth holes, and 50 Î¼l of the standard dilution was added to the ninth and tenth holes, respectively, and 50 Î¼l of each of the ninth and tenth holes was discarded. (The amount of each well was 50 Î¼l after dilution, and the concentrations were 90 ng/L, 60 ng/L, 30 ng/L, 15 ng/L, 7.5 ng/L, respectively). 2. Adding samples: Set blank holes separately (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Add 40 Î¼l of the sample dilution to the sample well to be tested on the enzyme-labeled plate, and then add 10 Î¼l of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix. 3. Incubation: The plate was sealed with a sealing film and incubated at 37 Â° C for 30 minutes. 4. Solution: 30 (48 times of 20T) concentrated washing solution was diluted with distilled water 30 (20 times of 48T) and used. 5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry. 6. Add enzyme: Add 50 Î¼l of enzyme labeling reagent to each well, except for blank wells. 7. Incubation: operation is the same as 3. 8. Washing: operation is the same as 5. 9. Color development: add 50 Î¼l of color developer A, add 50 Î¼l of color developer B, gently shake and mix, and avoid light at 37 Â°C. 15 minutes. 10. Termination: 50 Î¼l of stop solution was added to each well to stop the reaction (the blue color turned yellow). 11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution. Note: 1. The kit should be taken out from the refrigerated environment and allowed to equilibrate for 15-30 minutes at room temperature. If the enzyme label is unsealed after unsealing, the strip should be stored in a sealed bag. 2. Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing does not affect the result. 3. The sampler should be used for each step and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun. 4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple (Ã—nÃ—5). 5. The sealing film is intended for single use only to avoid cross-contamination. 6. Keep the substrate away from light. 7. Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading. 8. All samples, washings and various wastes should be treated as infectious materials. 9. The different batch components of this reagent must not be mixed. 10. In the case of an English manual, the English manual shall prevail. Calculation: taking the concentration of the standard as the abscissa and the OD as the ordinate, drawing a standard curve on the coordinate paper, and finding the corresponding concentration from the standard curve according to the OD value of the sample; multiplying by the dilution factor; or using the standard Calculate the linear regression equation of the standard curve by the concentration and OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample. (This figure is for reference only) Kit performance: 1. Sample linear regression and expected concentration correlation coefficient R value is 0.990 or more. 2. Within and within the batch should be less than 9% and 11% detection range: 5 ng / L -100 ng / L Storage conditions and expiration date: 1. Kit storage:; 2-8 Â° C. 2. Validity: 6 months
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